Qatar Univ. Sci. Bull. (1988), 8: 231-243
_EFFECT OF ORAL ADMINISTRATION OF ALCOHOL ON THE.
DEVELOPING RAT EMBRYOS
By
HAMZA AHMAD EL-SHABAKA
Department of Zoology, Faculty of Science, Qatar University, Doha
State of Qatar
Keywords: Alcohol affect. developing rat embroys.
ABSTRACT
Pregnant rats were forced to ingest 0.015 ml of 25% ethanol/gm body weight daily
starting from day 7 (D7-group) or day 9 (D9-group) until day 15 of gestation. The
number of surviving embryos significantly "decreased and more embryos were
resorbed in alcohol-treated groups. The mean weight of ethanol-and saline-treated
surviving embryos did not differ significantly. Alcohol administration in the
D7-group produced exencephaly and hydrocephaly while in D9-group only
hydrocephalic abnormality occurred. Alcohol treatment in both groups produced
destruction mainly in the neuroepithelium of the cerebral cortex. Numerous
degenerating cells and necrotic fragments were observed in the cerebral cortex of
D7-group. Moreover, the cytoplasm of the neuroepithelial cells of cerebral cortex
of D7-and D9-groups was highly vacuolated.
INTRODUCTION
The morphological teratogenicity of alcohol has been well documented in humans
and other experimental animals. Maternal alcohol consumption in humans results
in a group of abnormalities known as the fetal alcohol syndrome "FAS" (Jones and
Smith, 1973). The characteristic features of this syndrome are pre-and postnatal
growth retardation, facial dysmorphology, central nervous abnormalities and
skeletal and cardiovascular anomalies (Jones and Smith, 1973, 1975; Clarren and
Smith, 1978). Little (1977) and Hanson et al. (1978) have shown that consumption
of 350 mg/kg of absolute alcohol per day is sufficient to produce offspring with
FAS.
The effects of ethanol have been studied extensively during recent years in embryos
of rats and mice (Brown et al., 1979; Randall and Taylor, 1979; Sulik et al, 1981;
Hannigan and Burke, 1982; Sulik and Johnston, 1983; Webster et al, 1983;
Nakatsuji and Johnson, 1984; Sanchis et al., 1987), chick (Sandor, 1968),
Rat embryos, alcohol effect
amphibians (Naktsuji, 1983) and in monkey offspring (Scott and Fradkin, 1984;
Clarren et al. , 1987).
The timing, duration, dose and route of alcohol administration and the species or
strain used may influence the resulting effects (Borges and Lewis, 1981). Among
the previous literature, the dose and the route of alcohol administration in mice
vary from one intraperitoneal (IP) injection (Hannigan & Burke, 1982), two IP
injections, 4 or 6 hours apart (Sulik & Johnston, 1983; Webster et al., 1983;
Nakatsuji & Johnson, 1984), one or two oral doses, 4 hours apart (Webster et al.,
1983) and in diet from gestation day 5 through gestation day 10 (Randall & Tylor,
1979). However, the effect of a daily administration of ethanol on the developing
rat embryos has not been investigated.
Drinkers do not intake alcohol intermittently but usually in continuous manner.
Hence, the aim of the present investigation is to study the effect of daily alcohol
administration via the oral route on the developing rat embryos.
MATERIAL AND METHODS
Virgin female rats weighing 250-300 gm were placed with males (one male for three
females) for two hours in the early morning. The presence of sperms in the vaginal
smear at the end of this period indicated that mating had occurred and was referred
to as 0 day 0 hour of gestation.
Twenty five% ethanol in normal saline solution was given in a single da~ly dose of
2.9 mg/gm body weight (0.015 ml/gm of body weight), by intragastric intubation.
Alcohol administration was initiated on day 7 (D7-group) or day 9 (D9-group) of
pregnant rats and continued through day 15 of gestation. Pregnant control rats
were orally intubated daily with 0.015 ml/gm body weight of normal saline solution.
Alcohol-treated and control pregnant rats were sacrificed on days 12 and 15 of
gestation by cervical dislocation, and the uteri were removed and placed in a
normal saline solution. Embryos were freed from the uterine tissue and the number
of surviving embryos was counted. Survivors were weighed, examined for external
malformations and then fixed in Zenker's or Bouin's fluids. Fixed embryos were
then dehydrated, cleared, embedded in paraffin, serially sectioned at 6 microns and
stained with hematoxylin and eosin. The significance of the differences between
alcohol-treated and control embryos were tested using the Student's t-test.
RESULTS
The mean number of survivors during the course of the experiment is reported in
Tables 1 and 2. The number of surviving embryos was smaller in alcohol-treated
mothers on gestational day 12 in both D7-(p<0.02) and D9-(p<0.05) groups and
during day 15 in both D7-(p<0.01) and D9(p<0.02) groups than in the
232
HAMZA AHMAD EL-SHABAKA
Table 1
The effect of acute alcohol treatment at day 7 and continued through day 15 of
gestation on the developing rat embryos.
Embryos at day 12 of
gestation
Observations
control
Embryos at day 15 of
gestation
alcohol-treated
control
:t 0.636
7.4 ± 0.562
(p<0.02)
10.6 ± 0.634
6.8 ± 0.814
(p<0.01)
Mean number of
resorbed ± S.E.
0.0 ± 0.000
2.8 ± 0.374
(p<0.001)
0.2 ± 0.245
4.0 ± 0.707
(p<0.001)
Mean body weight
(gm) ± S.E.
0.025 ± 0.002
0.022 ± 0.001
(p>0.2)
0.173 ± 0.021
0.209 ± 0.006
(p>0.2)
120.0 ± 7.949
102.75 ± 8.220
(P>0.2)
150.0 ± 10.946
138.0 ± 5.336
(p>0.4)
Mean number of
survivors ± S.E.
9.8
Mean thickness of '
cerebral cortex
(microns)±S.E.
lj
alcohol-treated
Table 2
The effect of acute alcohol treatment at da,y 9 and continued through day 15 of
gestation on the developing rat embryos.
Observations
Embryos at day 12 of
gestation
Embryos at day 15 of
gestation
control
alcohol-treated
control
Mean number of
survivors±S.E.
10.4±0.678
8.2±0.583
(P 0.05)
10.80.583
7.4±±0.927
(P 0.02)
Mean number of
resorbed±S.E.
0.2±0.2
1.6±0.4
(p 0.02)
0.00±0.00
2.2±0.583
(p 0.01)
Mean body weight
(gm) ± S.E.
0.025±0.002
0.022±0.001
(p 0.2)
0.170±0.014
0.190±0.005
(p 0.2)
121.0±6.347
104.8±7.696
(P>0.2)
151.0±11.539
146.0±10.689
(p>0.9)
Mean thickness of
cerebral cortex
(microns)±S.E.
233
alcohol-treated
Rat embryos, alcohol effect
corresponding control groups. Also a significant resorption incidence was observed
.on days 12 and 15 of gestation in both 07- and 09-groups (Tables 1 and 2).
On the other ~slight changes, but insignificant, in the embryo body weigJtt
(excluding resorptions) were observed in both 07- and 09- groups (Tables 1 and
2).
Incidence of Malformations
On day 12 of gestation, the developing 07- and 09-embryos (14 and 82 embryos
respectively) showed no morphological malformations, except thaf:tbe forebrains
of some embryos were somewhat narrower and smaller than those of the
saline-treated controls (Figs, 1,2 and 3). After 15 days of ges~atiop, eight embryos
(11.76%) showed exencephally (Fig. 5) and 3 embryos (4.41%) showed hydrocephaly in the 07-group. In the 09-group, 11 embryos (14.86%) showed
hydrocephaly (Fig. 6) but no exencephalic abnormalities were detected. On the
other hand, the saline-treated controls showed no incidence of morphological
abnormalities.
HISTOLOGY
1-12-day embryos
In saline-treated embryos, beginning either after 7 ot 9 days of gestation, the
cerebral cortex of the brain consisted of stratified epithelial cells each with an oval
nucleus containing one or more nucleoli. The nuclei of the inner ependymal layer
showed highly mitotic figures (Fig. 7). The apical borders of the neuroepithelium
presented the normal ruffled appearance due to the numerous pseudopodia!
processes (Fig. 7).
In alcohol treated 07-group, an insignificant decrease in the cerebral cortical
thickness occurred (Table 1). The neuroepithelium consisted of stratified epithelial
cells containing irregularly shaped and darkly stained nuclei. Numerous vacuoles
were also visible in the cytoplasm of other neuroepithelial cells. Moreover,
numerous cells showed different stages of degeneration with many darkly stained
particles suggestive of nuclear and cytoplasmic debris of these cells. In many places,
the apical border oJ the neuroepithelium was more or less smooth with no
pseudopodia! processes (Fig. 8).
In the 09-group an insignificant decrease of the cerebral cortex thickness was
shown in day 12 of gestation (table 2). The neuroepithelium exhibited a normal
appearance except that the cells were less dense and contained numerous vacuoles.
The mitotic figures in the ependymal layer as well as the pseudopodia! processes of
the apical border were less numerous as compared with those of the corresponding
controls (Fig. 9).
234
HAMZA AHMAQ EL-SHABAKA
1
Fig. 1-3:
Rat embryos on day 12 of gestation showing the heads of control (Fig.
1), D7-embryo with small prosencephalon (arrow) (Fig. 2) and
D9-embryo with reduced prosencephalon (arrow) (Fig. 3)
235
Rat embryos, alcohol effect
Figs. 4-6:
Rat embryos on day 15 of gestation showing the heads of control (Fig.
4), D7-embryo showing exencephaly (Fig. 5) and D9-embryo with
hydrocephaly (Fig. 6).
236
HAMZA AHMAD EL-SHABAKA
2-15-day embryos
On day 15, the ependymal layer of the cerebral cortex of control embryos
proliferated actively and the neuroepithelium appeared much thicker and
contained more cell layres. The nuclei of the inner layer, the ependymal layer,
show high mitotic figures (Fig. 10).
In the D7-group, a few necrotic particles were still present and the mitotic figures
were very scant. The cerebral cortex decreased slightly in thickness (Table I), but
the neuroepithelial cells contained oval nuclei which may be considered a sign of
repairing. In addition, numerous small vacuoles were still visible in the cytoplasm
of the cerebral cortex. The intercellular spaces of the neuroepithelium were more
enlarged than those of the control embryos (Fig. 11).
In the 09-group, the cerebral cortex slightly decreased in thickness (Table II) as
compared with that of corresponding control. The neuroepithelial cells contained
small rounded nuclei, and many vacuoles were observed in the cytoplasm of these
cells especially near the inner limiting membrane. The mitotic figures in the
ependymal cell layer were fewer (Fig. 12) than in corresponding control.
DISCUSSION
In the present study, a daily dose of alcohol closely resembling that used by the
average alcoholic drinker was given orally to the pregnant rats beginning with 7 or 9
days after gestation. Alcohol administration caused a higher embryo mortality than
in controls. Similar results were obtained in mice embryos (Randall and Taylor,
1979; Bannigan and Burke, 1982; Webster et al., 1983).
A large number of rat embryos was resorbed in ·alcohol-treated embryos than in
controls. Similar results were obtained in mice embryos (Randall and Taylor, 1979;
Bannigan and Burke, 1982; Webster et al., 1983). Moreover, the frequency and
severity of resorption incidence were greater in the D7-group than in the
D9~embryos. This may be due to a higher sensitivity of early embryonic stages to
alcohol and/or to the long term exposure of D7-group to the. alcohol. Also, Clarren
et al. (1987) reported that administration of ethanol orally once per week to gravid
Macaca nemestrina increased the rate of spontaneous abortion at and above
1-8grnlkg body weight. Hence, daily drinking alcohol by pregnant women,
especially at early stage and before being aware of pregnancy, may induce
abortion.
The result of the pr.esent investigation showed no significant changes in the embryo
body weight after alcohol administration. This result confirms the findin~~ reported
by Kronick(1976), Randall and Taylor (1979) and Bannigan and Burke (1982). On
the other hand, Scott and Fradkin (1984) found that birthweight of cynomolgus
monkeys Macaca fascicularis was significantly lowered-when the mothers received 5
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Figs. 7-9: Transverse sections through the fore-brains of developing rat etnbryos on gestation day 12 of control
(Fig. 7), D7-etnbryos (Fig. 8) and D9-embryo (Fig. 9) showing the cytoplasmic vacuoles (arrows) and
necrotic particles (arrowheads). X 700
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Figs. 10-12: Transverse sections through the fore-brains of developing rat embryos on gestation day 15 of
control (Fig. 10), D7-embryos (Fig. 11) and D9-embryo (Fig. 12) showing the cytoplasmic
vacuoles (arrows) and necrotic particles (arrowheads). X 700
Rat embryos, alcohol effect
gmlkg/day of ethanol by intragastric intubation from day 20 to 150 of gestation.
Alcohol intake also produced some morphological abnormalities in the head region
including reduction of forebrain, hydrocephaly (in D7-and D9-embryos) and
exencephaly (in D7-group). The variation in the type of malformations with the day
of administration suggests that certain organogenic stages of the embryos may be
more sensitive to alcohol than other stages. On the other hand, no evidence of
facial defects was observed in any of the ethanol exposed embryos. This result
confirms that of Scott and Fradkin (1984) in cynomolgus monkeys. Also, pregnant
mice fed a chocolate-flavored liquid diet provided with ethanol from gestation day
5 through gestation day 10 produced limb and eye defects, hydrocephaly and
exencephaly but no facial abnormalities were noted (Randall and Taylor, 1979).
However, the typical midfacial abnormalities could be produced in mice by a brief
period of maternal alcohol intoxication during gastrulation (Sulik et al., 1981; Sulik
& Lauder, 1983). Moreover, two IP injections,4 or 6 hours apart, of 25% ethanol
on gestation day 7 produced eye abnormalities, deficient derivatives of medial nasal
prominences, exencephaly (Sulik & Johnston, 1983), mandibular and maxillary
hypoplasia, narrow face and/or absence of mandible (Webster et al., 1983). The
latter authors also reported that two IP injections, 4 or 6 hours apart, of alcohol on
gestational day 9 produced mainly defects of limbs besides a few cases of
exencephaly, omphalocoeles and facial defects. Ther.outeofalcohol administration
rather than the amount of alcohol administered or the stage of embryonic
development at the time of exposure may be critical factor in producing facial
defects in the developing rat embryos. This hypothesis is under investigation.
Administration of alcohol to pregnant rats produced extensive cellular damage of
the cerebral cortex with no alteration of the neuroepithelial thickness. However,
Bannigan and Burke (1982) reported retardation of the neuroepithelium of
alcohol-treated embryos. The presence of cytoplasmic vacuoles in D7-and
D9-g7oups suggests that ethyl alcohol may be toxic to the cells, affecting the
cytoP.lasm or the cell membrane of the neuroepithelium (Hannigan and Burke,
1982). Moreover, ethanol treatment caused susceptible effect for cell divisions of
ependymal layer. These results suggest that a long exposure period of early rat
embryos can produce destructive effects of the cerebral cortex.
On gestation day 15, some neuroepithelial cells showed signs of repairing. The
capacity of repairing was demonstrated by the smaller number and size of vacuoles
and cellular debris. However, the intercellular spaces were still large as compared
with those in saline-treated embryos. Hannigan and Burke (1982) reported that,
fifty hours after alcohol injection, the neuroepithelium was completely cleared of
debris which resulted from cell necrosis. They also suggested that the intercellular
spaces may not have been caused directly by ethanol but might be due to sodium
240
HAMZA AHMAD EL-SHABAKA
retention. Further physiological and cytological studies are required to explain the
mechanisms of alcohol cytotoxicity to the neuroepithelium.
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Rat embryos, alcohol effect
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242
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